Project Summary/Abstract RNA viruses are among the most rapidly evolving species. As a consequence, they readily escape host immunity and acquire resistance to antivirals. To combat these viruses we must utilize strategies that account for their evolutionary flexibility. We propose to study the molecular mechanisms of RNA virus evolution, in particular hepatitis C virus (HCV), to inform the development of `evolution proof' strategies for vaccine and antiviral drug design. To this end, we will examine how constraints on protein structure and function, in the form of epistasis, shape virus evolution. Our approach is to map networks of epistatically interacting residues within viral proteins through statistical analysis of coevolution and direct detection of epistasis using a state-of- the-art technique for population sequencing. With these maps, we will examine the flexibility of viral protein constraints and their impact on the capacity and direction of virus evolution. Furthermore, though ultra-deep sequencing of HCV populations treated with panels of antiviral drugs, we will define landscapes of antiviral resistance in order to systematically explore the contribution of epistasis to patterns of evolution. By examining a major source of protein constraint and the evolutionary mechanisms that facilitate bypass of those constraints, thus enabling viral adaptation, this study will provide new insights into key facets of the evolutionary process that may precipitate novel strategies for targeting rapidly evolving viral pathogens.